Anti-alpha v beta 3 recombinant human antibodies, nucleic acids encoding same and methods of use

ABSTRACT

The invention provides a LM609 grafted antibody exhibiting selective binding affinity to α v β 3 . The LM609 grafted antibody consists of at least one LM609 CDR grafted heavy chain polypeptide and at least one LM609 CDR grafted light chain polypeptide or functional fragment thereof. Nucleic acids encoding LM609 grafted heavy and light chains as well as nucleic acids encoding the parental non-human antibody LM609 are additionally provided. Functional fragments of such encoding nucleic acids are similarly provided. The invention also provides a method of inhibiting a function of α v β 3 . The method consists of contacting α v β 3  with a LM609 grafted antibody or functional fragment thereof under conditions which allow binding to α v β 3 . Finally, the invention provides for a method of treating an α v β 3 -mediated disease. The method consists of administering an effective amount of a LM609 grafted antibody or functional fragment thereof under conditions which allow binding to α v β 3 .

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to integrin mediateddiseases and, more particularly, to nucleic acids encodingα_(v)β₃-inhibitory monoclonal antibodies and to CDR graftedα_(v)β₃-inhibitory antibodies for the therapeutic treatment ofα_(v)β₃-mediated diseases.

[0002] Integrins are a class of cell adhesion receptors that mediateboth cell-cell and cell-extracellular matrix adhesion events. Integrinsconsist of heterodimeric polypeptides where a single α chain polypeptidenoncovalently associates with a single β chain. There are now about 14distinct α chain polypeptides and at least about 8 different β chainpolypeptides which constitute the integrin family of cell adhesionreceptors. In general, different binding specificities and tissuedistributions are derived from unique combinations of the α and β chainpolypeptides or integrin subunits. The family to which a particularintegrin is associated with is usually characterized by the β subunit.However, the ligand binding activity of the integrin is largelyinfluenced by the a subunit. For example, vitronectin binding integrinscontain the α_(v) integrin subunit.

[0003] It is now known that the vitronectin binding integrins consist ofat least three different α_(v) containing integrins. These α_(v)containing integrins include α_(v)β₃, α_(v)β₁ and α_(v)β₅, all of whichexhibit different ligand binding specificities. For example, in additionto vitronectin, α_(v)β₃ binds to a large variety of extracellular matrixproteins including fibronectin, fibrinogen, laminin, thrombospondin, vonWillebrand factor, collagen, osteospontin and bone sialoprotein I. Theintegrin α_(v)β₁ binds to fibronectin, osteopontin and vitronectinwhereas α_(v)β₅ is known to bind to vitronectin and osteopontin.

[0004] As cell adhesion receptors, integrins are involved in a varietyof physiological processes including, for example, cell attachment, cellmigration and cell proliferation. Different integrins play differentroles in each of these biological processes and the inappropriateregulation of their function or activity can lead to variouspathological conditions. For example, inappropriate endothelial cellproliferation during neovascularization of a tumor has been found to bemediated by cells expressing vitronectin binding integrins. In thisregard, the inhibition of the vitronectin-binding integrin α_(v)β₃ alsoinhibits this process of tumor neovascularization. By this samecriteria, α_(v)β₃ has also been shown to mediate the abnormal cellproliferation associated with restenosis and granulation tissuedevelopment in cutaneous wounds, for example. Additional diseases orpathological states mediated or influenced by α_(v)β₃ include, forexample, metastasis, osteoporosis, age-related macular degeneration anddiabetic retinopathy, and inflammatory diseases such as rheumatoidarthritis and psoriasis. Thus, agents which can specifically inhibitvitronectin-binding integrins would be valuable for the therapeutictreatment of diseases.

[0005] Many integrins mediate their cell adhesive functions byrecognizing the tripeptide sequence Arg-Gly-Asp (RGD) found within alarge number of extracellular matrix proteins. A variety of approacheshave attempted to model agents after this sequence to target aparticular integrin-mediated pathology. Such approaches include, forexample, the use of RGD-containing peptides and peptide analogues whichrely on specificity to be conferred by the sequences flanking the RGDcore tripeptide sequence. Although there has been some limited success,most RGD-based inhibitors have been shown to be, at most, selective forthe targeted integrin and therefore exhibit some cross-reactivity toother non-targeted integrins. Such cross-reactive inhibitors thereforelack the specificity required for use as an efficacious therapeutic.This is particularly true for previously identified inhibitors of theintegrin α_(v)β₃.

[0006] Monoclonal antibodies on the other hand exhibit the specificityrequired to be used as an effective therapeutic. Antibodies also havethe advantage in that they can be routinely generated againstessentially any desired antigen. Moreover, with the development ofcombinatorial libraries, antibodies can now be produced faster and moreefficiently than by previously used methods within the art. The use ofcombinatorial methodology also allows for the selection of the desiredantibody along with the simultaneous isolation of the encoding heavy andlight chain nucleic acids. Thus, further modification can be performedto the combinatorial antibody without the incorporation of an additionalcloning step.

[0007] Regardless of the potential advantages associated with the use ofmonoclonal antibodies as therapeutics, these molecules nevertheless havethe drawback in that they are almost exclusively derived from non-humanmammalian organisms. Therefore, their use as therapeutics is limited bythe fact that they will normally elicit a host immune response. Methodsfor substituting the antigen binding site or complementarity determiningregions (CDRs) of the non-human antibody into a human framework havebeen described. Such methods vary in terms of which amino acid residuesshould be substituted as the CDR as well as which framework residuesshould be changed to maintain binding specificity. In this regard, it isunderstood that proper orientation of the β sheet architecture, correctpacking of the heavy and light chain interface and appropriateconformation of the CDRs are all important for preserving antigenspecificity and affinity within the grafted antibody. However, all ofthese methods require knowledge of the nucleotide and amino acidsequence of the non-human antibody and the availability of anappropriately modeled human framework.

[0008] Thus, there exists a need for the availability of nucleic acidsencoding integrin inhibitory antibodies which can be used as compatibletherapeutics in humans. For α_(v)β₃-mediated diseases, the presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0009] The invention provides a LM609 grafted antibody exhibitingselective binding affinity to α_(v)β₃. The LM609 grafted antibodyconsists of at least one LM609 CDR grafted heavy chain polypeptide andat least one LM609 CDR grafted light chain polypeptide or functionalfragment thereof. Nucleic acids encoding LM609 grafted heavy and lightchains as well as nucleic acids encoding the parental non-human antibodyLM609 are additionally provided. Functional fragments of such encodingnucleic acids are similarly provided. The invention also provides amethod of inhibiting a function of α_(v)β₃. The method consists ofcontacting α_(v)β₃ with a LM609 grafted antibody or functional fragmentthereof under conditions which allow binding to α_(v)β₃. Finally, theinvention provides for a method of treating an α_(v)β₃-mediated disease.The method consists of administering an effective amount of a LM609grafted antibody or functional fragment thereof under conditions whichallow binding to α_(v)β₃.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows the nucleotide and deduced amino acid sequence of thevariable region of the LM609 grafted antibody. FIG. 1A shows thenucleotide and deduced amino acid sequences for the LM609 grafted heavychain variable region (Gln1-Ser117; SEQ ID NOS:1 and 2,respectively)while FIG. 1B shows the nucleotide and deduced amino acid sequences forthe LM609 grafted light chain variable region (Glu1-Lys107; SEQ ID NOS:3and 4, respectively).

[0011]FIG. 2 shows the nucleotide and deduced amino acid sequence of thevariable region of the monoclonal antibody LM609. FIG. 2A shows thenucleotide and deduced amino acid sequence of the LM609 heavy chainvarible region (SEQ ID NOS:5 and 6, respectively). The variable regionextends from amino acid Glu1 to Ala117. FIG. 2B shows the nucleotide anddeduced amino acid sequence of the LM609 light chain variable region(SEQ ID NOS:7 and 8, respectively). The variable region of the lightchain extends from amino acid Asp1 to Lys107.

[0012]FIG. 3 shows the competitive inhibition of LM609 IgG binding tothe integrin α_(v)β₃ with recombinant LM609 Fab. Soluble recombinantmurine LM609 Fab fragments were prepared from periplasmic fractions ofM13 bacteriophage clones muLM609M13 12 and muLM609M13 29. The periplasmsamples were serially diluted, mixed with either 1 ng/ml, 5 ng/ml, or 50ng/ml of LM609 IgG and then incubated in 96 well plates coated withpurified α_(v)β₃. Plates were washed and bound LM609 IgG detected withgoat anti-murine Fc specific antibody conjugated to alkalinephosphatase. Fab produced by clone muLM609M13 12 inhibits both 1 ng/mland 5 ng/ml LM609 IgG binding at all concentrations of Fab greater than1:27 dilution.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention is directed to nucleic acids encoding themonoclonal antibody (MAb) LM609. This antibody specifically recognizesthe integrin α_(v)β₃ and inhibits its functional activity. The inventionis also directed to nucleic acids encoding and to polypeptidescomprising non-murine forms of LM609 termed LM609 grafted antibodies. ALM609 grafted antibody retains the binding specificity and inhibitoryactivity of its parent murine antibody LM609.

[0014] In one embodiment, the hybridoma expressing LM609 was used as asource to generate and clone cDNAs encoding LM609. The heavy and lightchain encoding cDNAs were sequenced and their CDR regions as defined byKabat et al., supra were substituted into a human antibody framework togenerate the non-murine form of the antibody. As an antibody having CDRsgrafted to a human acceptor framework, it is unlikely that LM609 graftedantibodies will elicit a host immune response and can therefore beadvantageously used for the treatment of α_(v)β₃-mediated diseases.

[0015] As used herein, the term “monoclonal antibody LM609” or “LM609”is intended to mean the murine monoclonal antibody specific for theintegrin α_(v)β₃ which is described by Cheresh, D. A. Proc. Natl. Acad.Sci. USA 84:6471-6475 (1987) and by Cheresh and Spiro J. Biol. Chem.262:17703-17711 (1987). LM609 was produced against and is reactive withthe M21 cell adhesion receptor now known as the integrin α_(v)β₃. LM609inhibits the attachment of M21 cells to α_(v)β₃ ligands such asvitronectin, fibrinogen and von Willebrand factor (Cheresh and Spiro,supra) and is also an inhibitor of α_(v)β₃-mediated pathologies such astumor induced angiogenesis (Brooks et al. Cell 79:1157-1164 (1994),granulation tissue development in cutaneous wound (Clark et al., Am. J.Pathology, 148:1407-1421 (1996)) and smooth muscle cell migration suchas that occurring during restenosis (Choi et al., J. Vascular Surg.,19:125-134 (1994); Jones et al., Proc. Natl. Acad. Sci. 93:2482-2487(1996)).

[0016] As used herein, the term “LM609 grafted antibody” is intended torefer to a non-mouse antibody or functional fragment thereof havingsubstantially the same heavy and light chain CDR amino acid sequences asfound in LM609 and absent of the substitution of LM609 amino acidresidues outside of the CDRs as defined by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983). The term “LM609 grafted antibody” or “LM609 grafted”when used in reference to heavy or light chain polypeptides is intendedto refer to a non-mouse heavy or light chain or functional fragmentthereof having substantially the same heavy or light chain CDR aminoacid sequences as found in the heavy or light chain of LM609,respectively, and also absent of the substitution of LM609 residuesoutside of the CDRs as defined by Kabat et al., supra. When used inreference to a functional fragment, not all LM609 CDRs need to berepresented. Rather, only those CDRs that would normally be present inthe antibody portion that corresponds to the functional fragment areintended to be referenced as the LM609 CDR amino acid sequences in theLM609 grafted functional fragment. Similarly, the term “LM609 graftedantibody” or “LM609 grafted” used in reference to an encoding nucleicacid is intended to refer to a nucleic acid encoding a non-mouseantibody or functional fragment being absent of the substitution ofLM609 amino acids outside of the CDRs as defined by Kabat et al., supraand having substantially the same nucleotide sequence as the heavy andlight chain CDR nucleotide sequences and encoding substantially the sameCDR amino acid sequences as found in LM609 and as defined by Kabat etal., supra.

[0017] The term “grafted antibody” or “grafted” when used in referenceto heavy or light chain polypeptides or functional fragments thereof isintended to refer to a heavy or light chain or functional fragmentthereof having substantially the same heavy or light chain of a donorantibody, respectively, and also absent of the substitution of donoramino acid residues outside of the CDRs as defined by Kabat et al.,supra. When used in reference to a functional fragment, not all donorCDRs need to be represented. Rather, only those CDRs that would normallybe present in the antibody portion that corresponds to the functionalfragment are intended to be referenced as the donor CDR amino acidsequences in the functional fragment. Similarly, the term “graftedantibody” or “grafted” when used in reference to an encoding nucleicacid is intended to refer to a nucleic acid encoding an antibody orfunctional fragment, being absent of the substitution of donor aminoacids outside of the CDRs as defined by Kabat et al., supra and havingsubstantially the same nucleotide sequence as the heavy and light chainCDR nucleotide sequences and encoding substantially the same CDR aminoacid sequences as found in the donor antibody and as defined by Kabat etal., supra.

[0018] The meaning of the above terms are intended to include minorvariations and modifications of the antibody so long as its functionremains uncompromised. Functional fragments such as Fab, F(ab)₂, Fv,single chain Fv (scFv) and the like are similarly included within thedefinition of the terms LM609 and LM609 grafted antibody. Suchfunctional fragments are well known to those skilled in the art.Accordingly, the use of these terms in describing functional fragmentsof LM609 or LM609 grafted antibodies are intended to correspond to thedefinitions well known to those skilled in the art. Such terms aredescribed in, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biologyand Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.),New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics,22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515(1989) and in Day, E. D., Advanced Immunochemistry, Second Ed.,Wiley-Liss, Inc., New York, N.Y. (1990).

[0019] As with the above terms used for describing functional fragmentsof LM609 and a LM609 grafted antibody, the use of terms which referenceother LM609, or LM609 grafted antibody domains, functional fragments,regions, nucleotide and amino acid sequences and polypeptides orpeptides, is similarly intended to fall within the scope of the meaningof each term as it is known and used within the art. Such terms include,for example, “heavy chain polypeptide” or “heavy chain”, “light chainpolypeptide” or “light chain”, “heavy chain variable region” (V_(H)) and“light chain variable region” (V_(L)) as well as the term“complementarity determining region” (CDR).

[0020] In the case where there are two or more definitions of a termwhich is used and/or accepted within the art, the definition of the termas used herein is intended to include all such meanings unlessexplicitly stated to the contrary. A specific example is the use of theterm “CDR” to describe the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.This particular region has been described by Kabat et al., supra, and byChothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum etal., J. Mol. Biol. 262:732-745 (1996) where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof LM609, LM609 grafted antibodies or variants thereof is intended to bewithin the scope of the term as defined and used herein. The amino acidresidues which encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. TABLE 1 CDRDefinitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101  93-101 V_(L)CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L) CDR3 89-9791-96 89-96

[0021] As used herein, the term “substantially” or “substantially thesame” when used in reference to a nucleotide or amino acid sequence isintended to mean that the nucleotide or amino acid sequence shows aconsiderable degree, amount or extent of sequence identity when comparedto a reference sequence. Such considerable degree, amount or extent ofsequence identity is further considered to be significant and meaningfuland therefore exhibit characteristics which are definitivelyrecognizable or known. Thus, a nucleotide sequence which issubstantially the same nucleotide sequence as a heavy or light chain ofLM609, or a LM609 grafted antibody including fragments thereof, refersto a sequence which exhibits characteristics that are definitively knownor recognizable as encoding or as being the amino acid sequence of LM609or a LM609 grafted antibody. Minor modifications thereof are included solong as they are recognizable as a LM609 or a LM609 grafted antibodysequence. Similarly, an amino acid sequence which is substantially thesame amino acid sequence as a heavy or light chain of LM609 graftedantibody or functional fragment thereof, refers to a sequence whichexhibits characteristics that are definitively known or recognizable asrepresenting the amino acid sequence of a LM609 grafted antibody andminor modifications thereof.

[0022] As used herein, the term “fragment” when used in reference to anucleic acid encoding LM609 or a LM609 grafted antibody is intended tomean a nucleic acid having substantially the same sequence as a portionof a nucleic acid encoding LM609 or a LM609 grafted antibody. Thenucleic acid fragment is sufficient in length and sequence toselectively hybridize to a LM609 or a LM609 grafted antibody encodingnucleic acid or a nucleotide sequence that is complementary to an LM609or LM609 grafted antibody encoding nucleic acid. Therefore, fragment isintended to include primers for sequencing and polymerase chain reaction(PCR) as well as probes for nucleic acid blot or solution hybridization.The meaning of the term is also intended to include regions ofnucleotide sequences that do not directly encode LM609 polypeptides suchas the introns, and the untranslated region sequences of the LM609encoding gene.

[0023] As used herein, the term “functional fragment” when used inreference to a LM609 grafted antibody or to heavy or light chainpolypeptides thereof is intended to refer to a portion of a LM609grafted antibody including heavy or light chain polypeptides which stillretains some or all or the α_(v)β₃ binding activity, α_(v)β₃ bindingspecificity and/or integrin α_(v)β₃-inhibitory activity. Such functionalfragments can include, for example, antibody functional fragments suchas Fab, F(ab)₂, Fv, single chain Fv (scFv). Other functional fragmentscan include, for example, heavy or light chain polypeptides, variableregion polypeptides or CDR polypeptides or portions thereof so long assuch functional fragments retain binding activity, specificity orinhibitory activity. The term is also intended to include polypeptidesencompassing, for example, modified forms of naturally occurring aminoacids such as D-stereoisomers, non-naturally occurring amino acids,amino acid analogues and mimetics so long as such polypeptides retainfunctional activity as defined above.

[0024] The invention provides a nucleic acid encoding a heavy chainpolypeptide for a LM609 grafted antibody or a functional fragmentthereof. Also provided is a nucleic acid encoding a light chainpolypeptide for a LM609 grafted antibody or a functional fragmentthereof. The nucleic acids consist of substantially the same heavy orlight chain variable region nucleotide sequences as those shown in FIGS.1A and 1B (SEQ ID NOS:1 and 3, respectively) or a fragment thereof.

[0025] A LM609 grafted antibody, including functional fragments thereof,is a non-mouse antibody which exhibits substantially the same bindingactivity, binding specificity and inhibitory activity as LM609. TheLM609 grafted antibody Fv fragments described herein are produced byfunctionally replacing CDRs as defined by Kabat et al., hereinafterreferred to as “Kabat CDRs,” within human heavy and light chain variableregion polypeptides with the Kabat CDRs derived from LM609. Functionalreplacement of the CDRs was performed by recombinant methods known tothose skilled in the art. Such methods are commonly referred to as CDRgrafting and are the subject matter of U.S. Pat. No. 5,225,539. Suchmethods can also be found described in “Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man,” Clark,M. (ed.), Nottingham, England: Academic Titles (1993).

[0026] Substitution of amino acid residues outside of the Kabat CDRs canadditionally be performed to maintain or augment beneficial bindingproperties of LM609 grafted antibodies so long as such amino acidsubstitutions do not correspond to a donor amino acid at that particularposition. Such substitutions allow for the modulation of bindingproperties without imparting any mouse sequence characteristics onto theantibody outside of the Kabat CDRs. Although the production of suchantibodies is described herein with reference to LM609 graftedantibodies, the substitution of such non-donor amino acids outside ofthe Kabat CDRs can be utilized for the production of essentially anygrafted antibody. The production of LM609 grafted antibodies isdescribed further below in Example II.

[0027] Briefly, LM609 nucleic acid fragments having substantially thesame nucleotide and encoding substantially the same amino acid sequenceof each of the heavy and light chain CDRs were synthesized andsubstituted into each of the respective human chain encoding nucleicacids. Modifications were performed within the non-Kabat CDR frameworkregion. These individual changes were made by generating a population ofKabat CDR grafted heavy and light chain variable regions wherein allpossible non-donor amino acid changes outside of the Kabat CDRs wererepresented and then selecting the appropriate antibody by screening thepopulation for binding activity. This screen resulted in the selectionof the LM609 grafted antibodies described herein.

[0028] The nucleotide sequences of the LM609 grafted heavy and lightchain variable regions are shown in FIGS. 1A and 1B, respectively. Thesesequences correspond substantially to those that encode the heavy andlight chain variable region polypeptides of a LM609 grafted antibody.These nucleic acids are intended to include both the sense andanti-sense strands of the LM609 grafted antibody encoding sequences.Single- and double-stranded nucleic acids are similarly included as wellas non-coding portions of the nucleic acid such as introns, 5′- and3′-untranslated regions and regulatory sequences of the gene forexample.

[0029] As shown in FIG. 1A, the LM609 grafted heavy chain variableregion polypeptide is encoded by a nucleic acid of about 351 nucleotidesin length which begins at the amino terminal Gln1 residue of thevariable region through to Ser117. This heavy chain variable regionencoding nucleic acid is joined to a human IgG1 constant region to yielda coding region of 1431 nucleotides which encodes a heavy chainpolypeptide of 477 total amino acids. Shown in FIG. 1B is the LM609grafted light chain variable region polypeptide which is encoded by anucleic acid of about 321 nucleotides in length beginning at the aminoterminal Glu1 residue of the variable region through to Lys107. Thislight chain variable region nucleic acid is joined to a human kappaconstruct region to yield a coding region of 642 nucleotides which codefor a light chain polypeptide of 214 total amino acids.

[0030] Minor modification of these nucleotide sequences are intended tobe included as LM609 grafted heavy and light chain encoding nucleicacids and their functional fragments. Such minor modifications include,for example, those which do not change the encoded amino acid sequencedue to the degeneracy of the genetic code as well as those which resultin only a conservative substitution of the encoded amino acid sequence.Conservative substitutions of encoded amino acids include, for example,amino acids which belong within the following groups: (1) non-polaramino acids (Gly, Ala, Val, Leu, and Ile); (2) polar neutral amino acids(Cys, Met, Ser, Thr, Asn, and Gln); (3) polar acidic amino acids (Aspand Glu); (4) polar basic amino acids (Lys, Arg and His); and (5)aromatic amino acids (Phe, Trp, Tyr, and His). Other minor modificationsare included within the nucleic acids encoding LM609 grafted heavy andlight chain polypeptides so long as the nucleic acid or encodedpolypeptides retain some or all of their function as described herein.

[0031] Thus, the invention also provides a nucleic acid encoding a LM609grafted heavy chain or functional fragment thereof wherein the nucleicacid encodes substantially the same LM609 grafted heavy chain variableregion amino acid sequence as that shown in FIG. 1A (SEQ ID NO:2) or afragment thereof. Similarly, the invention also provides a nucleic acidencoding a LM609 grafted light chain or functional fragment thereofwherein the nucleic acid encodes substantially the same light chainvariable region amino acid sequence as that shown in FIG. 1B (SEQ IDNO:4) or a fragment thereof.

[0032] In addition to conservative substitutions of amino acids, minormodifications of the LM609 grafted antibody encoding nucleotidesequences which allow for the functional replacement of amino acids arealso intended to be included within the definition of the term. Thesubstitution of functionally equivalent amino acids encoded by the LM609grafted antibody nucleotide sequences is routine and can be accomplishedby methods known to those skilled in the art. Briefly, the substitutionof functionally equivalent amino acids can be made by identifying theamino acids which are desired to be changed, incorporating the changesinto the encoding nucleic acid and then determining the function of therecombinantly expressed and modified LM609 grafted polypeptide orpolypeptides. Rapid methods for making and screening multiplesimultaneous changes are well known within the art and can be used toproduce a library of encoding nucleic acids which contain all possibleor all desired changes and then expressing and screening the library forLM609 grafted polypeptides which retain function. Such methods include,for example, codon based mutagenesis, random oligonucleotide synthesisand partially degenerate oligonucleotide synthesis.

[0033] Codon based mutagenesis is the subject matter of U.S. Pat. Nos.5,264,563 and 5,523,388 and is advantageous for the above proceduressince it allows for the production of essentially any and all desiredfrequencies of encoded amino acid residues at any and all particularcodon positions within an oligonucleotide. Such desired frequenciesinclude, for example, the truly random incorporation of all twenty aminoacids or a specified subset thereof as well as the incorporation of apredetermined bias of one or more particular amino acids so as toincorporate a higher or lower frequency of the biased residues comparedto other incorporated amino acid residues. Random oligonucleotidesynthesis and partially degenerate oligonucleotide synthesis cansimilarly be used for producing and screening for functionallyequivalent amino acid changes. However, due to the degeneracy of thegenetic code, such methods will incorporate redundancies at a desiredamino acid position. Random oligonucleotide synthesis is the coupling ofall four nucleotides at each nucleotide position within a codon whereaspartially degenerate oligonucleotide synthesis is the coupling of equalportions of all four nucleotides at the first two nucleotide positions,for example, and equal portions of two nucleotides at the thirdposition. Both of these latter synthesis methods can be found describedin, for example, Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382,(1990) and Devlin et al., Science 249:404-406, (1990).

[0034] Identification of amino acids to be changed can be accomplishedby those skilled in the art using current information availableregarding the structure and function of antibodies as well as availableand current information encompassing methods for CDR graftingprocedures.

[0035] Using the above described methods known within the art, any orall of the non-identical amino acids can be changed either alone or incombination with amino acids at different positions to incorporate thedesired number of amino acid substitutions at each of the desiredpositions. The LM609 grafted polypeptides containing the desiredsubstituted amino acids are then produced and screened for retention oraugmentation of function compared to the unsubstituted LM609 graftedpolypeptides. Production of the substituted LM609 grafted polypeptidescan be accomplished by, for example, recombinant expression usingmethods known to those skilled in the art. Those LM609 graftedpolypeptides which exhibit retention or augmentation of functioncompared to unsubstituted LM609 grafted polypeptides are considered tocontain minor modifications of the encoding nucleotide sequence whichresult in the functional replacement of one or more amino acids.

[0036] The functional replacement of amino acids is beneficial whenproducing grafted antibodies having human framework sequences since itallows for the rapid identification of equivalent amino acid residueswithout the need for structural information or the laborious proceduresnecessary to assess and identify which amino acid residues should beconsidered for substitution in order to successfully transfer bindingfunction from the donor. Moreover, it eliminates the actual step-wiseprocedures to change and test the amino acids identified forsubstitution. Essentially, using the functional replacement approachdescribed above, all non-identical amino acid residues between the donorand the human framework can be identified and substituted with any orall other possible amino acid residues, excluding the correspondingdonor amino acid, at each non-identical position to produce a populationof substituted polypeptides containing all possible or all desiredpermutations and combinations. The population of substitutedpolypeptides can then be screened for those substituted polypeptideswhich retain function. Using the codon based mutagenesis proceduresdescribed above, the generation of a library of substituted amino acidresidues and the screening of functionally replaced residues has beenused for the rapid production of grafted therapeutic antibodies as wellas for the rapid alteration of antibody affinity. Such procedures areexemplified in, for example, Rosok et al., J. Biol. Chem.271:22611-22618 (1996) and in Glaser et al., J. Immunol. 149:3903-3913(1992), respectively.

[0037] The invention further provides fragments of LM609 grafted heavyand light chain encoding nucleic acids wherein such fragments consistsubstantially of the same nucleotide or amino acid sequence as the LM609grafted variable region of the heavy or light chain polypeptides. Thevariable region of the heavy chain polypeptide consists essentially ofnucleotides 1-351 and of amino acid residues Gln1 to Ser117 of FIG. 1A(SEQ ID NOS:1 and 2, respectively). The variable region of the lightchain polypeptide consists essentially of nucleotides 1-321 and of aminoacid residues Glu1 to Lys107 of FIG. 1B (SEQ ID NOS:3 and 4,respectively). The termini of such variable region encoding nucleicacids is not critical so long as the intended purpose and functionremains the same.

[0038] Fragments additional to the variable region nucleic acidfragments are provided as well. Such fragments include, for example,nucleic acids consisting substantially of the same nucleotide sequenceas a CDR of a LM609 grafted heavy or light chain polypeptide. Sequencescorresponding to the LM609 grafted CDRs include, for example, thoseregions defined by Kabat et al., supra, and/or those regions defined byChothia et al., supra, as well as those defined by MacCallum et al.,supra. The LM609 grafted CDR fragments for each of the above definitionscorrespond to the nucleotides set forth below in Table 2. The nucleotidesequence numbering is taken from the primary sequence shown in FIGS. 1Aand 1B (SEQ ID NOS:1 and 3) and conforms to the definitions previouslyset forth in Table 1. TABLE 2 LM609 Grafted CDR Nucleotide ResiduesKabat Chothia MacCallum V_(H) CDR1  91-105 76-96  88-105 V_(H) CDR2148-198 157-168 139-177 V_(H) CDR3 295-318 298-315 289-315 V_(L) CDR1 70-102 76-96  88-108 V_(L) CDR2 148-168 148-156 136-165 V_(L) CDR3265-291 271-288 265-288

[0039] Similarly, the LM609 grafted CDR fragments for each of the abovedefinitions correspond to the amino acid residues set forth below inTable 3. The amino acid residue number is taken from the primarysequence shown in FIGS. 1A and 1B (SEQ ID NOS:2 and 4) and conforms tothe definitions previously set forth in Table 1. TABLE 3 LM609 GraftedCDR Amino Acid Residues Kabat Chothia MacCallum V_(H) CDR1 Ser31—Ser35Gly26—Tyr32 Ser30—Ser35 V_(H) CDR2 Lys50—Gly66 Ser53—Gly56 Trp47—Tyr59V_(H) CDR3 His99—Tyr106 Asn100—Ala105 Ala97—Ala105 V_(L) CDR1Gln24—His34 Ser26—His32 Ser30—Tyr36 V_(L) CDR2 Tyr50—Ser56 Tyr50—Ser52Leu46—Ile55 V_(L) CDR3 Gln89—Thr97 Ser91—His96 Gln89—His96

[0040] Thus, the invention also provides nucleic acid fragments encodingsubstantially the same amino acid sequence as a CDR of a LM609 graftedheavy or light chain polypeptide.

[0041] Nucleic acids encoding LM609 grafted heavy and light chainpolypeptides and fragments thereof are useful for a variety ofdiagnostic and therapeutic purposes. For example, the LM609 graftednucleic acids can be used to produce LM609 grafted antibodies andfunctional fragments thereof having binding specificity and inhibitoryactivity against the integrin α_(v)β₃. The antibody and functionalfragments thereof can be used for the diagnosis or therapeutic treatmentof α_(v)β₃-mediated disease. A LM609 grafted antibody and functionalfragments thereof can be used, for example, to inhibit binding activityor other functional activities of α_(v)β₃ that are necessary forprogression of an α_(v)β₃-mediated disease. Other functional activitiesnecessary for progression of α_(v)β₃-mediated disease include, forexample, the activation of α_(v)β₃, α_(v)β₃-mediated signal transductionand the α_(v)β₃-mediated prevention of apoptosis. Advantageously,however, a LM609 grafted antibody comprises non-mouse framework aminoacid sequences and as such is less antigenic in regard to the inductionof a host immune response. The LM609 grafted antibody nucleic acids ofthe invention can also be used to model functional equivalents of theencoded heavy and light chain polypeptides.

[0042] Thus, the invention provides LM609 grafted heavy chain and LM609grafted light chain polypeptides or functional fragments thereof. TheLM609 grafted heavy chain polypeptide exhibits substantially the sameamino acid sequence as that shown in FIG. 1A (SEQ ID NO:2) or functionalfragment thereof whereas the LM609 grafted light chain polypeptideexhibits substantially the same amino acid sequence as that shown inFIG. 1B (SEQ ID NO:4) or functional fragment thereof. Also provided is aLM609 grafted antibody or functional fragment thereof. The antibody isgenerated from the above heavy and light chain polypeptides orfunctional fragments thereof and exhibits selective binding affinity toα_(v)β₃.

[0043] The invention provides a nucleic acid encoding a heavy chainpolypeptide for monoclonal antibody LM609 or functional fragmentthereof. Also provided is a nucleic acid encoding a light chainpolypeptide for monoclonal antibody LM609 or a functional fragmentthereof. The nucleic acids consist of substantially the same heavy orlight chain variable region nucleotide sequences as that shown in FIGS.2A and 2B (SEQ ID NOS:5 and 7, respectively) or a fragment thereof.

[0044] As described previously, monoclonal antibody LM609 has been shownin the art to have binding activity to the integrin α_(v)β₃. Althoughspecificity can in principle be generated towards essentially anytarget, LM609 is an integrin inhibitory antibody that exhibitssubstantial specificity and inhibitory activity to a single memberwithin an integrin family. In this case, LM609 exhibits substantialspecificity and inhibitory activity to the α_(v)β₃ integrin within theβ₃ family. The amino acid or nucleotide sequence of monoclonal antibodyLM609 has never been previously isolated and characterized.

[0045] The isolation and characterization of LM609 encoding nucleicacids was performed by techniques known to those skilled in the art andwhich are described further below in the Examples. Briefly, cDNA fromhybridoma LM609 was generated and used as the source for which toisolate LM609 encoding nucleic acids. Isolation was performed by firstdetermining the N-terminal amino acid sequence for each of the heavy andlight chain polypeptides and then amplifying by PCR the antibodyencoding sequences from the cDNA. The 5′ primers were reverse translatedto correspond to the newly determined N-terminal amino acid sequenceswhereas the 3′ primers corresponded to sequences substantially similarto antibody constant region sequences. Amplification and cloning of theproducts resulted in the isolation of the nucleic acids encoding heavyand light chains of LM609.

[0046] The nucleotide sequences of the LM609 heavy and light chainvariable region sequences are shown in FIGS. 2A and 2B, respectively.These sequences correspond substantially to those that encode thevariable region heavy and light chain polypeptides of LM609. As with theLM609 grafted antibody nucleic acids, these LM609 nucleic acids areintended to include both sense and anti-sense strands of the LM609encoding sequences. Single- and double-stranded nucleic acids are alsoincluded as well as non-coding portions of the nucleic acid such asintrons, 5′- and 3′-untranslated regions and regulatory sequences of thegene for example.

[0047] As shown in FIG. 2A, the LM609 heavy chain variable regionpolypeptide is encoded by a nucleic acid of about 351 nucleotides inlength which begins at the amino terminal Glu1 residue of the variableregion through to Ala 117. The murine LM609 antibody heavy chain has anIgG2a constant region. Shown in FIG. 2B is the LM609 light chainvariable region polypeptide which is encoded by a nucleic acid of about321 nucleotides in length which begins at the amino terminal Asp1residue of the variable region through to Lys 107. In the functionalantibody, LM609 has a kappa light chain constant region.

[0048] As with the LM609 grafted antibody nucleic acids, minormodifications of these LM609 nucleotide sequences are intended to beincluded as heavy and light chain LM609 encoding nucleic acids. Suchminor modifications are included within the nucleic acids encoding LM609heavy and light chain polypeptides so long as the nucleic acids orencoded polypeptides retain some or all of their function as described.

[0049] Thus, the invention also provides a nucleic acid encoding a LM609heavy chain or functional fragment wherein the nucleic acid encodessubstantially the same variable region amino acid sequence of monoclonalantibody LM609 as that shown in FIG. 2A (SEQ ID NO:6) or a fragmentthereof. Similarly, the invention also provides a nucleic acid encodinga LM609 light chain or functional fragment wherein the nucleic acidencodes substantially the same variable region amino acid sequence ofmonoclonal antibody LM609 as that shown in FIG. 2B (SEQ ID NO:8) or afragment thereof.

[0050] The invention further provides fragments of LM609 heavy and lightchain encoding nucleic acids wherein such fragments consistsubstantially of the same nucleotide or amino acid sequence as thevariable region of LM609 heavy or light chain polypeptides. The variableregion of the LM609 heavy chain polypeptide consists essentially ofnucleotides 1-351 and of amino acid residues Glu1 to Ala117 of FIG. 2A(SEQ ID NOS:5 and 6, respectively). The variable region of the LM609light chain polypeptide consists essentially of nucleotides 1-321 and ofamino acid residues Asp1 to lys107 of FIG. 2B (SEQ ID NOS:7 and 8,respectively). The termini of such variable region encoding nucleicacids is not critical so long as the intended purpose and functionremains the same. Such intended purposes and functions include, forexample, use for the production of recombinant polypeptides or ashybridization probes for heavy and light chain variable regionsequences.

[0051] Fragments additional to the variable region nucleic acidfragments are provided as well. Such fragments include, for example,nucleic acids consisting substantially of the same nucleotide sequenceas a CDR of a LM609 heavy or light chain polypeptide. Sequencescorresponding to the LM609 CDRs include, for example, those regionswithin the variable region which are defined by Kabat et al., supra,and/or those regions within the variable regions which are defined byChothia et al., supra, as well as those regions defined by MacCallum etal., supra. The LM609 CDR fragments for each of the above definitionscorrespond to the nucleotides set forth below in Table 4. The nucleotidesequence numbering is taken from the primary sequence shown in FIGS. 2Aand 2B (SEQ ID NOS:5 and 7) and conforms to the definitions previouslyset forth in Table 1. TABLE 4 LM609 CDR Nucleotide Residues KabatChothia MacCallum V_(H) CDR1  91-105 76-96  88-105 V_(H) CDR2 148-198157-168 139-177 V_(H) CDR3 295-318 298-315 289-315 V_(L) CDR1  70-10276-96  88-108 V_(L) CDR2 148-168 148-156 136-165 V_(L) CDR3 265-291271-288 265-288

[0052] Similarly, the LM609 fragments of each of the above definitionscorrespond to the amino acid residues set forth below in Table 5. Theamino acid residue numbering is taken from the primary sequence shown inFIGS. 2A and 2B (SEQ ID NOS:6 and 8) and conforms to the definitions setforth in Table 1. TABLE 5 LM609 CDR Amino Acid Residues Kabat ChothiaMacCallum V_(H) CDR1 Ser31—Ser35 Gly26—Tyr32 Ser30—Ser35 V_(H) CDR2Lys50—Gly66 Ser53—Gly56 Trp47—Tyr59 V_(H) CDR3 His99—Tyr106Asn100—Ala105 Ala97—Ala105 V_(L) CDR1 Gln24—His34 Ser26—His32Ser30—Tyr36 V_(L) CDR2 Tyr50—Ser56 Tyr50—Ser52 Leu46—Ile55 V_(L) CDR3Gln89—Thr97 Ser91—His96 Gln89—His96

[0053] Nucleic acids encoding LM609 heavy and light chain polypeptidesand fragments thereof are useful for a variety of diagnostic andtherapeutic purposes. For example, the LM609 nucleic acids can be usedto produce recombinant LM609 antibodies and functional fragments thereofhaving binding specificity and inhibitory activity against the integrinα_(v)β₃. The antibody and functional fragments thereof can be used todetermine the presence or absence of α_(v)β₃ in a sample to diagnose thesusceptibility or occurrence of an α_(v)β₃-mediated disease.Alternatively, the recombinant LM609 antibodies and functional fragmentsthereof can be used for the therapeutic treatment of α_(v)β₃-mediateddiseases or pathological state. As with a LM609 grafted antibody,recombinant LM609 and functional fragments thereof can be used toinhibit the binding activity or other functional activities of α_(v)β₃that are necessary for progression of the α_(v)β₃-mediated disease orpathological state.

[0054] The LM609 nucleic acids of the invention can also be used tomodel functional equivalents of the encoded heavy and light chainpolypeptides. Such functional equivalents can include, for example,synthetic analogues or mimics of the encoded polypeptides or functionalfragments thereof. A specific example would include peptide mimetics ofthe LM609 CDRs that retain some or substantially the same binding orinhibitory activity of LM609. Additionally, the LM609 encoding nucleicacids can be used to engineer and produce nucleic acids which encodemodified forms or derivatives of the antibody LM609, its heavy and lightchain polypeptides and functional fragments thereof. As describedpreviously, such modified forms or derivatives include, for example,non-mouse antibodies, their corresponding heavy and light chainpolypeptides and functional fragments thereof which exhibitsubstantially the same binding and inhibitory activity as LM609.

[0055] The invention also provides a method of treating anα_(v)β₃-mediated disease. The method consists of administering aneffective amount of a LM609 grafted antibody or a functional fragmentthereof under conditions which allow binding to α_(v)β₃. Also providedis a method of inhibiting a function of α_(v)β₃. The method consists ofcontacting α_(v)β₃ with a LM609 grafted antibody or a functionalfragment thereof under conditions which allow binding to α_(v)β₃.

[0056] As described previously, a LM609 grafted antibody is a monoclonalantibody which exhibits essentially all of the binding characteristicsas does its parental CDR-donor antibody LM609. These characteristicsinclude, for example, significant binding specificity and affinity forthe integrin α_(v)β₃. The Examples below demonstrate these bindingproperties and further show that the binding of such antibodies toα_(v)β₃ inhibits α_(v)β₃ ligand binding and function. Thus, LM609grafted antibodies are useful for a large variety of diagnostic andtherapeutic purposes directed to the inhibition of α_(v)β₃ function.

[0057] The integrin α_(v)β₃ functions in numerous cell adhesion andmigration associated events. As such, the dysfunction or dysregulationof this integrin, its function, or of cells expressing this integrin, isassociated with a large number of diseases and pathological conditions.The inhibition α_(v)β₃ binding or function can therefore be used totreat or reduce the severity of such α_(v)β₃-mediated pathologicalconditions. Described below are examples of several pathologicalconditions mediated by α_(v)β₃, since the inhibition of at least thisintegrin reduces the severity of the condition. These examples areintended to be representative and as such are not inclusive of allα_(v)β₃-mediated diseases. For example, there are numerous pathologicalconditions additional to those discussed below which exhibit thedysregulation of α_(v)β₃ binding, function or the dysregulation of cellsexpressing this integrin and in which the pathological condition can bereduced, or will be found to be reduced, by inhibiting the bindingα_(v)β₃. Such pathological conditions which exhibit this criteria, areintended to be included within the definition of the term as usedherein.

[0058] Angiogenesis, or neovascularization, is the process where newblood vessels form from pre-existing vessels within a tissue. Asdescribed further below, this process is mediated by endothelial cellsexpressing α_(v)β₃ and inhibition of at least this integrin, inhibitsnew vessel growth. There are a variety of pathological conditions thatrequire new blood vessel formation or tissue neovascularization andinhibition of this process inhibits the pathological condition. As such,pathological conditions that require neovascularization for growth ormaintenance are considered to be α_(v)β₃-mediated diseases. The extentof treatment, or reduction in severity, of these diseases will thereforedepend on the extent of inhibition of neovascularization. Theseα_(v)β₃-mediated diseases include, for example, inflammatory disorderssuch as immune and non-immune inflammation, chronic articularrheumatism, psoriasis, disorders associated with inappropriate orinopportune invasion of vessels such as diabetic retinopathy,neovascular glaucoma and capillary proliferation in atheroscleroticplaques as well as cancer disorders. Such cancer disorders can include,for example, solid tumors, tumor metastasis, angiofibromas, retrolental,fibroplasia, hemangiomas, Kaposi's sarcoma and other cancers whichrequire neovascularization to support tumor growth. Additional diseaseswhich are considered angiogenic include psoriasis and rheumatoidarthritis as well as retinal diseases such as macular degeneration.Diseases other than those requiring new blood vessels which areα_(v)β₃-mediated diseases include, for example, restenosis andosteoporosis.

[0059] Treatment of the α_(v)β₃-mediated diseases can be performed byadministering an effective amount of a LM609 grafted antibody or afunctional fragment thereof so as to bind to α_(v)β₃ and inhibit itsfunction. Administration can be performed using a variety of methodsknown in the art. The choice of method will depend on the specificα_(v)β₃-mediated disease and can include, for example, the in vivo, insitu and ex vivo administration of a LM609 grafted antibody orfunctional fragment thereof, to cells, tissues, organs, and organisms.Moreover, such antibodies or functional fragments can be administered toan individual exhibiting or at risk of exhibiting an α_(v)β₃-mediateddisease. Definite clinical diagnosis of an α_(v)β₃-mediated diseasewarrants the administration of a LM609 grafted antibody or a functionalfragment thereof. Prophylactic applications are warranted in diseaseswhere the α_(v)β₃-mediated disease mechanisms precede the onset of overtclinical disease. Thus, individuals with familial history of disease andpredicted to be at risk by reliable prognostic indicators can be treatedprophylactically to interdict α_(v)β₃-mediated mechanisms prior to theironset.

[0060] LM609 grafted antibody or functional fragments thereof can beadministered in a variety of formulations and pharmaceuticallyacceptable media for the effective treatment or reduction in theseverity of an α_(v)β₃-mediated disease. Such formulations andpharmaceutically acceptable medias are well known to those skilled inthe art. Additionally, a LM609 grafted antibody or functional fragmentsthereof can be administered with other compositions which can enhance orsupplement the treatment or reduction in severity of an α_(v)β₃-mediateddisease. For example, the coadministration of a LM609 grafted antibodyto inhibit tumor-induced neovascularization and a chemotherapeutic drugto directly inhibit tumor growth is one specific case where theadministration of other compositions can enhance or supplement thetreatment of an α_(v)β₃-mediated disease.

[0061] A LM609 grafted antibody or functional fragments are administeredby conventional methods, in dosages which are sufficient to cause theinhibition of α_(v)β₃ integrin binding at the sight of the pathology.Inhibition can be measured by a variety of methods known in the art suchas in situ immunohistochemistry for the prevalence of α_(v)β₃ containingcells at the site of the pathology as well as include, for example, theobserved reduction in the severity of the symptoms of theα_(v)β₃-mediated disease.

[0062] In vivo modes of administration can include intraperitoneal,intravenous and subcutaneous administration of a LM609 grafted antibodyor a functional fragment thereof. Dosages for antibody therapeutics areknown or can be routinely determined by those skilled in the art. Forexample, such dosages are typically administered so as to achieve aplasma concentration from about 0.01 μg/ml to about 100 μg/ml,preferably about 1-5 μg/ml and more preferably about 5 μg/ml. In termsof amount per body weight, these dosages typically correspond to about0.1-300 mg/kg, preferably about 0.2-200 mg/kg and more preferably about0.5-20 mg/kg. Depending on the need, dosages can be administered once ormultiple times over the course of the treatment. Generally, the dosagewill vary with the age, condition, sex and extent of theα_(v)β₃-mediated pathology of the subject and should not be so high asto cause adverse side effects. Moreover, dosages can also be modulatedby the physician during the course of the treatment to either enhancethe treatment or reduce the potential development of side effects. Suchprocedures are known and routinely performed by those skilled in theart.

[0063] The specificity and inhibitory activity of LM609 graftedantibodies and functional fragments thereof allow for the therapeutictreatment of numerous α_(v)β₃-mediated diseases. Such diseases include,for example, pathological conditions requiring neovascularization suchas tumor growth, and psoriasis as well as those directly mediated byα_(v)β₃ such as restenosis and osteoporosis. Thus, the inventionprovides methods and LM609 grafted antibody containing compositions forthe treatment of such diseases.

[0064] Throughout this application various publications are referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

[0065] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Isolation and Characterization of LM609 Encoding Nucleic Acids

[0066] This Example shows the cloning and sequence determination ofLM609 encoding nucleic acids.

[0067] LM609 is directed against the human vitronectin receptor,integrin α_(v)β₃. α_(v)β₃ is highly upregulated in melanoma,glioblastoma, and mammary carcinoma and plays a role in theproliferation of M21 melanoma cells both in vitro and in vivo. α_(v)β₃also plays a role in angiogenesis, restenosis and the formation ofgranulation tissue in cutaneous wounds. LM609 has been shown to inhibitthe adhesion of M21 cells to vitronectin as well as preventproliferation of M21 cells in vitro. Thus, grafting of LM609 couldresult in a clinically valuable therapeutic agent.

[0068] cDNA Synthesis of LM609 Variable Regions: For cDNA synthesis,total RNA was prepared from 10⁸ LM609 hybridoma cells using amodification of the method described by Chomczynski and Sacchi(Chomczynski and Sacchi, Analyt. Biochem. 162:156 (1987)). LM609variable (V) region genes were cloned by reversetranscription-polymerase chain reaction (RT-PCR) and cDNA wassynthesized using BRL Superscript kit. Briefly, 5 μg of total cellularRNA, 650 ng oligo dT and H₂O were brought to a total volume of 55 μl.The sample was heated to 70° C. for 10 min and chilled on ice. Reactionbuffer was added and the mixture brought to 10 mM DTT and 1 mM dNTPs andheated at 37° C. for 2 minutes. 5 μl (1000 units) reverse transcriptasewas added and incubated at 37° C. for 1 hour and then chilled on ice.

[0069] All oligonucleotides were synthesized by β-cyanoethylphosphoramidite chemistry on an ABI 394 DNA synthesizer.Oligonucleotides used for PCR amplification and routine site-directedmutagenesis were purified using oligonucleotide purification cartridges(Applied Biosystems, Foster City, Calif.). Forward PCR primers weredesigned from N-terminal protein sequence data generated from purifiedLM609 antibody. The forward PCR primers contained sequences coding forthe first six amino acids in each antibody variable chain (proteinsequenced at San Diego State University). The sequence of the lightchain forward PCR primer (997) was 5′-GCC CAA CCA GCC ATG GCC GAT ATTGTG CTA ACT CAG-3′ (SEQ ID NO:19) whereas the light chain reverse PCRprimer (734) was 5′-AC AGT TGG TGC AGC ATC AGC-3′ (SEQ ID NO:20) used.This reverse primer corresponds to mouse light chain kappa amino acidresidues 109-115. The sequence of the heavy chain forward PCR primer(998) was 5′-ACC CCT GTG GCA AAA GCC GAA GTG CAG CTG GTG GAG-3′ (SEQ IDNO:21). Heavy chain reverse PCR primer 733: 5′-GA TGG GGG TGT CGT TTTGGC-3′ SEQ ID NO:22). The PCR primers also contain regions of homologywith specific sequences within the immunoexpression vector.

[0070] V_(L) and V_(H) chains were amplified in two separate 50 μlreaction mixtures containing 2 μl of the cDNA-RNA heteroduplex, 66.6 mMTris-HCl pH 8.8, 1.5 mM MgCl₂, 0.2 mM of each four dNTPs, 10 mM2-mercaptoethanol, 0.25 units Taq polymerase (Boehringer-Mannheim,Indianapolis, Ind) and 50 pmoles each of primers 997 and 734 and 998 and733, respectively. The mixtures were overlaid with mineral oil andcycled for two rounds of PCR with each cycle consisting of 30 seconds at94° C. (denature), 30 seconds at 50° C. (anneal), and 30 seconds at 72°C. (synthesis). This reaction was immediately followed by 30 cycles ofPCR consisting of 30 seconds at 94° C. (denature), 30 seconds at 55° C.(anneal), and 30 seconds at 72° C. (synthesis) followed by a finalsynthesis reaction for 5 minutes at 72° C. The reaction products werepooled, extracted with CHCl₃ and ethanol precipitated.

[0071] Amplified products were resuspended in 20 μl TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 8.0) and electrophoresed on a 5% polyacrylamidegel. Bands migrating at expected molecular weights of V_(H) and V_(L)were excised, chemically eluted from the gel slice, extracted withorganic solvents and ethanol precipitated.

[0072] Cloning of amplified V_(H) and V_(L) genes into M13 phageimmunoexpression vector: The amplified V region gene products weresequentially cloned into the phage immunoexpression vector byhybridization mutagenesis (Near, R. Biotechniques 12:88 (1992); Yeltonet al., J. Immunol. 155:1994-2003 (1995)). Introduction of the amplifiedV_(L) and V_(H) sequences by hybridization mutagenesis positions theantibody sequences in frame with the regulatory elements contained inthe M13 vector required for efficient Fab expression. One advantage ofthis technique is that no restriction endonuclease sites need to beincorporated into the V_(L) or V_(H) gene sequences for cloning as isdone with conventional DNA ligation methods.

[0073] To perform the cloning, 400 ng each of the double-strandedamplified products were first phosphorylated with polynucleotide kinase.100 ng of the phosphorylated LM609 V_(L) product was mixed with 250 ngof uridinylated BS11 phage immunoexpression vector, denatured by heatingto 90° C. and annealed by gradual cooling to room temperature. BS11 isan M13 immunoexpression vector derived from M13 IX and encodes CH₁ ofmurine IgG1 and murine kappa light chain constant domain (Huse, W. D.In: Antibody Engineering: A Practical Guide, C. A. K. Borrebaeck, ed. W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).Nucleotide sequences included in the PCR amplification primers anneal tocomplementary sequences present in the single-stranded BS11 vector. Theannealed mixture was fully converted to a double-stranded molecule withT4 DNA polymerase plus dNTPs and ligated with T4 ligase. 1 μl of themutagenesis reaction was electroporated into E. coli strain DH10B,titered onto a lawn of XL-1 E. coli and incubated until plaques formed.Plaque lift assays were performed as described using goat anti-murinekappa chain antibody conjugated to alkaline phosphatase (Yelton et al,supra; Huse, W. D., supra). Fifteen murine light chain positive M13phage clones were isolated, pooled and used to prepare uridinylatedvector to serve as template for hybridization mutagenesis with the PCRamplified LM609 V_(H) product.

[0074] Clones expressing functional murine LM609 Fab were identified bybinding to purified α_(v)β₃ by ELISA. Briefly, Immulon II ELISA plateswere coated overnight with 1 μg/ml (100 ng/well) α_(v)β₃ and nonspecificsites blocked for two hours at 27° C. Soluble Fabs were prepared byisolating periplasmic fractions of cultures of E. coli strain MK30-3(Boehringer Mannheim Co.) infected with the Fab expressing M13 phageclones. Periplasm fractions were mixed with binding buffer 100 mM NaCl,50 mM Tris pH 7.4, 2mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 0.02% NaN₃, 1mg/ml BSA and incubated with immobilized α_(v)β₃ for two hours at 27° C.Plates were washed with binding buffer and bound Fab detected with goatanti-murine kappa chain antibody conjugated to alkaline phosphatase.Four α_(v)β₃ reactive clones were identified: muLM609M13 12, 29, 31 and69. MuLM609M13 12 and 29 gave the strongest signals in the ELISA assay.DNA sequence analysis showed that clones muLM609M13 12, 31 and 69 allhad identical light chain sequence and confirmed the previouslydetermined N-terminal amino acid sequence of purified LM609 light chainpolypeptide. All four clones had identical V_(H) DNA sequence and alsoconfirmed the previously determined N-terminal amino acid sequence ofpurified LM609 heavy chain polypeptide.

[0075] To further characterize the binding activity of each clone,soluble Fab fractions were prepared from 50 ml cultures of E. colistrain MK30-3 infected with clones 12 and 29 and evaluated for bindingto α_(v)β₃ in a competitive ELISA with LM609 IgG. The results of thisELISA are shown in FIG. 3. Clone muLM609M13 12 was found to inhibitLM609 IgG binding (at LM609 IgG concentrations of 1 ng/ml and 5 ng/ml)to α_(v)β₃ in a concentration dependent manner at periplasm titersranging from neat to 1:80. Clone muLM609M13 12 was plaque purified andboth the V region heavy and light chain DNA sequences again determined.Complete DNA sequence of the final clone, muLM609M13 12-5, is shown inFIGS. 2A and 2B.

EXAMPLE II Construction of LM609 Grafted Functional Antibody Fragments

[0076] This Example shows the construction of functional LM609 graftedantibody fragments in which only the CDRs have been transferred from theLM609 donor antibody to a human acceptor framework.

[0077] CDR grafting of LM609 to produce a functional antibody fragmentwas accomplished by the methods set forth below. These procedures areapplicable for the CDR grafting of essentially any donor antibody whereamino acid residues outside of the CDRs from the donor antibody are notdesired in the final grafted product.

[0078] Briefly, the protein sequence of the LM609 antibody, wasdetermined by cloning and sequencing the cDNA that encodes the variableregions of the heavy and light chains as described in Example I. TheCDRs from the LM609 donor antibody were identified and grafted intohomologous human variable regions of a human acceptor framework.Identification of CDR regions were based on the combination ofdefinitions published by Kabat et al., and MacCallum et al.

[0079] The boundaries of the CDR regions have been cumulatively definedby the above two publications and are residues 30-35, 47-66 and 97-106for CDRs 1, 2 and 3, respectively, of the heavy chain variable regionand residues 24-36, 46-56, and 89-97 for CDRs 1, 2 and 3, respectively,of the light chain variable region. Non-identical donor residues withinthese boundaries but outside of CDRs as defined by Kabat et al. wereidentified and were not substituted into the acceptor framework.Instead, functional non-donor amino acid residues were identified andsubstituted for certain of these non-identical residues.

[0080] As described below, the only non-identical residue outside of theCDRs as defined by Kabat et al. but within the CDRs as defined above isat position 49 of the LM609 light chain. To identify functionalnon-donor amino acids at this position, a library of nineteen antibodieswas constructed that contained all non-donor amino acids at position 49and then screened for binding activity against α_(v)β₃.

[0081] Human immunoglobulin sequences were identified from theBrookhaven Protein Data Bank-Kabat Sequences of Proteins ofImmunological Interest database (release 5.0). Human framework sequencesshowing significant identity to the murine LM609 variable region genesequences were selected for receiving the LM609 CDRS. Human heavy chainvariable region M72 'CL had 88% identity to frameworks 1, 2 and 3 ofLM609 heavy chain and human light chain V region LS1 'CL had 79%identity to frameworks 1, 2 and 3 of LM609 light chain. With theexclusion of non-identical residues outside of the CDRs as defined byKabat et al. murine LM609 CDR sequences as defined by Kabat et al. andMacCallum et al. were grafted onto the human frameworks. Using thisgrafting scheme, the final grafted product does not contain any aminoacid residues outside of the CDRs as defined by Kabat et al. which areidentical to an LM609 amino acid at the corresponding position (outsideof residues: 31-35, 50-66 and 99-106 for CDRs 1, 2 and 3, respectively,of the heavy chain variable region and residues 24-34, 50-56, and 89-97for CDRs 1, 2 and 3, respectively, of the light chain variable region).Moreover, no intermediates are produced which contain an amino acidresidue outside of the CDRs as defined by Kabat et al. which areidentical to the LM609 amino acid at that position. The CDR graftingprocedures are set forth below.

[0082] Full-length CDR grafted variable region genes were synthesized byPCR using long overlapping oligonucleotides. The heavy chainoligonucleotides map to the following nucleotide positions: V_(H)oligonucleotide 1 (V_(H) oligo1), nucleotides (nt) 1-84; (SEQ ID NO:9);V_(H)oligo2, nt 70-153, (SEQ ID NO:10); V_(H) oligo3, nt 138-225 (SEQ IDNO:11); V_(H) oligo4, nt 211-291 (SEQ ID NO:12); V_(H) oligo5, nt277-351 (SEQ ID NO:13).

[0083] The light chain variable region oligonucleotides were synthesizedso as to contain the CDR grafted variable region as well as a stopcondon at position 49. The five oligonucleotides for the light chainLM609 grafted variable region are shown as SEQ ID NOS:14-18 where thesecond oligonucleotide in the series contains the stop codon at position49 (SEQ ID NO:15).

[0084] All long oligonucleotides were gel purified. CDR grafting of theLM609 heavy chain variable region was constructed by mixing 5overlapping oligonucleotides (SEQ ID NOS:9-13), at equimolarconcentrations, in the presence of annealing PCR primers containing atleast 18 nucleotide residues complementary to vector sequences for theefficient annealing of the amplified V region product to thesingle-stranded vector. The annealed mixture was fully converted to adouble-stranded molecule with T4 DNA polymerase plus dNTPs and ligatedwith T4 ligase. The mutagenesis reaction (1 μl) was electroporated intoE. coli strain DH10B (BRL), titered onto a lawn of XL-1 (Stratagene,Inc.) and incubated until plaques formed. Replica filter lifts wereprepared and plaques containing V_(H) gene sequences were screenedeither by hybridization with a digoxigenin-labeled oligonucleotidecomplementary to LM609 heavy chain CDR 2 sequences or reactivity with7F11-alkaline phosphatase conjugate, a monoclonal antibody raisedagainst the decapeptide sequence Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser(SEQ ID NO:23) appended to the carboxy terminus of the vector CH₁ domain(Biosite, Inc., San Diego, Calif.).

[0085] Fifty clones that were double-positive were pooled and used toprepare uridinylated template for hybridization mutagenesis with theamplified CDR grafted LM609 V_(L) product constructed in a similarfashion using the five overlapping oligonucleotides shown as SEQ IDNOS:23-27. The mutagenesis reaction was electroporated into E. colistrain DH10B. Randomly picked clones were sequenced to identify aproperly constructed template for construction of the non-donor libraryat position 49. This template was prepared as a uridinylated templateand an oligonucleotide population of the following sequence was used forsite directed mutagenesis.

GGGAACGATA-19aa-GATGAGAAGC

[0086] The sequence 19aa in the above primer (SEQ ID NO:24) representsthe fact that this primer specifies a sequence population consisting of19 different codon sequences that encode each of the 19 non-donor aminoacids. These amino acids are those not found at position 49 of LM609 andinclude all amino acids except for Lys. Clones that resulted from thismutagenesis were picked and antibody expressed by these clones wereprepared. These samples were then screened for binding to α_(v)β₃ in anELISA assay. Clones having either Arg or Met amino acids in position 49were functionally identified. The nucleotide and amino acid sequence ofthe LM609 grafted heavy chain variable region is show in FIG. 1A (SEQ IDNOS:1 and 2, respectively). The nucleotide and amino acid sequence ofthe LM609 grafted light chain variable region is shown in FIG. 1B (SEQID NOS:3 and 4, respectively).

[0087] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 24 351 base pairs nucleic acid single linear not provided CDS 1..351 1CAG GTG CAG CTG GTG GAG TCT GGG GGA GGC GTT GTG CAG CCT GGA AGG 48 GlnVal Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGT AGC TAT 96 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 GACATG TCT TGG GTT CGC CAG GCT CCG GGC AAG GGT CTG GAG TGG GTC 144 Asp MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 GCA AAAGTT AGT AGT GGT GGT GGT AGC ACC TAC TAT TTA GAC ACT GTG 192 Ala Lys ValSer Ser Gly Gly Gly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 CAG GGC CGATTC ACC ATC TCC AGA GAC AAT AGT AAG AAC ACC CTA TAC 240 Gln Gly Arg PheThr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 CTG CAA ATGAAC TCT CTG AGA GCC GAG GAC ACA GCC GTG TAT TAC TGT 288 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 GCA AGA CAT AACTAC GGC AGT TTT GCT TAC TGG GGC CAA GGG ACT ACA 336 Ala Arg His Asn TyrGly Ser Phe Ala Tyr Trp Gly Gln Gly Thr Thr 100 105 110 GTG ACT GTT TCTAGT 351 Val Thr Val Ser Ser 115 117 amino acids amino acid linearprotein not provided 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val ValGln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Val 35 40 45 Ala Lys Val Ser Ser Gly Gly Gly Ser Thr TyrTyr Leu Asp Thr Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn SerLys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu AspThr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asn Tyr Gly Ser Phe Ala TyrTrp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 321 basepairs nucleic acid single linear not provided CDS 1..321 3 GAG ATT GTGCTA ACT CAG TCT CCA GCC ACC CTG TCT CTC AGC CCA GGA 48 Glu Ile Val LeuThr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GAA AGG GCGACT CTT TCC TGC CAG GCC AGC CAA AGT ATT AGC AAC CAC 96 Glu Arg Ala ThrLeu Ser Cys Gln Ala Ser Gln Ser Ile Ser Asn His 20 25 30 CTA CAC TGG TATCAA CAA AGG CCT GGT CAA GCC CCA AGG CTT CTC ATC 144 Leu His Trp Tyr GlnGln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 MKK TAT CGT TCC CAGTCC ATC TCT GGG ATC CCC GCC AGG TTC AGT GGC 192 Xaa Tyr Arg Ser Gln SerIle Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 AGT GGA TCA GGG ACA GATTTC ACC CTC ACT ATC TCC AGT CTG GAG CCT 240 Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 GAA GAT TTT GCA GTC TATTAC TGT CAA CAG AGT GGC AGC TGG CCT CAC 288 Glu Asp Phe Ala Val Tyr TyrCys Gln Gln Ser Gly Ser Trp Pro His 85 90 95 ACG TTC GGA GGG GGG ACC AAGGTG GAA ATT AAG 321 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105107 amino acids amino acid linear protein not provided 4 Glu Ile Val LeuThr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg AlaThr Leu Ser Cys Gln Ala Ser Gln Ser Ile Ser Asn His 20 25 30 Leu His TrpTyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Xaa Tyr ArgSer Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu AspPhe Ala Val Tyr Tyr Cys Gln Gln Ser Gly Ser Trp Pro His 85 90 95 Thr PheGly Gly Gly Thr Lys Val Glu Ile Lys 100 105 351 base pairs nucleic acidsingle linear not provided CDS 1..351 5 GAA GTG CAG CTG GTG GAG TCT GGGGGA GGC TTA GTG AAG CCT GGA AGG 48 Glu Val Gln Leu Val Glu Ser Gly GlyGly Leu Val Lys Pro Gly Arg 1 5 10 15 TCC CTG AGA CTC TCC TGT GCA GCCTCT GGA TTC GCT TTC AGT AGC TAT 96 Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Ala Phe Ser Ser Tyr 20 25 30 GAC ATG TCT TGG GTT CGC CAG ATT CCGGAG AAG AGG CTG GAG TGG GTC 144 Asp Met Ser Trp Val Arg Gln Ile Pro GluLys Arg Leu Glu Trp Val 35 40 45 GCA AAA GTT AGT AGT GGT GGT GGT AGC ACCTAC TAT TTA GAC ACT GTG 192 Ala Lys Val Ser Ser Gly Gly Gly Ser Thr TyrTyr Leu Asp Thr Val 50 55 60 CAG GGC CGA TTC ACC ATC TCC AGA GAC AAT GCCAAG AAC ACC CTA TAC 240 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala LysAsn Thr Leu Tyr 65 70 75 80 CTG CAA ATG AGC AGT CTG AAC TCT GAG GAC ACAGCC ATG TAT TAC TGT 288 Leu Gln Met Ser Ser Leu Asn Ser Glu Asp Thr AlaMet Tyr Tyr Cys 85 90 95 GCA AGA CAT AAC TAC GGC AGT TTT GCT TAC TGG GGCCAA GGG ACT CTG 336 Ala Arg His Asn Tyr Gly Ser Phe Ala Tyr Trp Gly GlnGly Thr Leu 100 105 110 GTC ACT GTC TCT GCA 351 Val Thr Val Ser Ala 115117 amino acids amino acid linear protein not provided 6 Glu Val Gln LeuVal Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met SerTrp Val Arg Gln Ile Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Lys ValSer Ser Gly Gly Gly Ser Thr Tyr Tyr Leu Asp Thr Val 50 55 60 Gln Gly ArgPhe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu GlnMet Ser Ser Leu Asn Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala ArgHis Asn Tyr Gly Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 ValThr Val Ser Ala 115 321 base pairs nucleic acid single linear notprovided CDS 1..321 7 GAT ATT GTG CTA ACT CAG TCT CCA GCC ACC CTG TCTGTG ACA CCA GGA 48 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser ValThr Pro Gly 1 5 10 15 GAT AGC GTC AGT CTT TCC TGC CAG GCC AGC CAA AGTATT AGC AAC CAC 96 Asp Ser Val Ser Leu Ser Cys Gln Ala Ser Gln Ser IleSer Asn His 20 25 30 CTA CAC TGG TAT CAA CAA AAA TCA CAT GAG TCT CCA AGGCTT CTC ATC 144 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg LeuLeu Ile 35 40 45 AAG TAT CGT TCC CAG TCC ATC TCT GGG ATC CCC TCC AGG TTCAGT GGC 192 Lys Tyr Arg Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe SerGly 50 55 60 AGT GGA TCA GGG ACA GAT TTC GCT CTC AGT ATC AAC AGT GTG GAGACT 240 Ser Gly Ser Gly Thr Asp Phe Ala Leu Ser Ile Asn Ser Val Glu Thr65 70 75 80 GAA GAT TTT GGA ATG TAT TTC TGT CAA CAG AGT GGC AGC TGG CCTCAC 288 Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Gly Ser Trp Pro His85 90 95 ACG TTC GGA GGG GGG ACC AAG CTG GAA ATT AAG 321 Thr Phe Gly GlyGly Thr Lys Leu Glu Ile Lys 100 105 107 amino acids amino acid linearprotein not provided 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu SerVal Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Gln Ala Ser GlnSer Ile Ser Asn His 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu SerPro Arg Leu Leu Ile 35 40 45 Lys Tyr Arg Ser Gln Ser Ile Ser Gly Ile ProSer Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Ser IleAsn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Met Tyr Phe Cys Gln GlnSer Gly Ser Trp Pro His 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu IleLys 100 105 84 base pairs nucleic acid single linear not provided 9CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTTGTGCAGC CTGGAAGGTC CCTGAGACTC 60TCCTGTGCAG CCTCTGGATT CACC 84 84 base pairs nucleic acid single linearnot provided 10 AACTTTTGCG ACCCACTCCA GACCCTTGCC CGGAGCCTGG CGAACCCAAGACATGTCATA 60 GCTACTGAAG GTGAATCCAG AGGC 84 87 base pairs nucleic acidsingle linear not provided 11 TGGGTCGCAA AAGTTAGTAG TGGTGGTGGTAGCACCTACT ATTTAGACAC TGTGCAGGGC 60 CGATTCACCA TCTCCAGAGA CAATAGT 87 81base pairs nucleic acid single linear not provided 12 TGCACAGTAATACACGGCTG TGTCCTCGGC TCTCAGAGAG TTCATTTGCA GGTATAGGGT 60 GTTCTTACTATTGTCTCTGG A 81 75 base pairs nucleic acid single linear not provided 13GTGTATTACT GTGCAAGACA TAACTACGGC AGTTTTGCTT ACTGGGGCCA AGGGACTACA 60GTGACTGTTT CTAGT 75 87 base pairs nucleic acid single linear notprovided 14 GAGATTGTGC TAACTCAGTC TCCAGCCACC CTGTCTCTCA GCCCAGGAGAAAGGGCGACT 60 CTTTCCTGCC AGGCCAGCCA AAGTATT 87 75 base pairs nucleicacid single linear not provided 15 TTAGATGAGA AGCCTTGGGG CTTGACCAGGCCTTTGTTGA TACCAGTGTA GGTGGTTGCT 60 AATACTTTGG CTGGC 75 84 base pairsnucleic acid single linear not provided 16 CCAAGGCTTC TCATCTAATATCGTTCCCAG TCCATCTCTG GGATCCCCGC CAGGTTCAGT 60 GGCAGTGGAT CAGGGACAGATTTC 84 81 base pairs nucleic acid single linear not provided 17GCTGCCACTC TGTTGACAGT AATAGACTGC AAAATCTTCA GGCTCCAGAC TGGAGATAGT 60GAGGGTGAAA TCTGTCCCTG A 81 57 base pairs nucleic acid single linear notprovided 18 CAACAGAGTG GCAGCTGGCC TCACACGTTC GGAGGGGGGA CCAAGGTGGAAATTAAG 57 36 base pairs nucleic acid single linear not provided 19GCCCAACCAG CCATGGCCGA TATTGTGCTA ACTCAG 36 20 base pairs nucleic acidsingle linear not provided 20 ACAGTTGGTG CAGCATCAGC 20 36 base pairsnucleic acid single linear not provided 21 ACCCCTGTGG CAAAAGCCGAAGTGCAGCTG GTGGAG 36 20 base pairs nucleic acid single linear notprovided 22 GATGGGGGTG TCGTTTTGGC 20 10 amino acids amino acid linearpeptide not provided 23 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 1 5 1023 base pairs nucleic acid single linear not provided misc_feature11..13 /note= “”NNN“ represents a codon specifying any amino acid otherthan Lys.” 24 GGGAACGATA NNNGATGAGA AGC 23

What is claimed is:
 1. A LM609 grafted antibody exhibiting selectivebinding affinity to α_(v)β₃ comprising at least one LM609 grafted heavychain polypeptide comprising substantially the same variable regionamino acid sequence as that shown in FIG. 1A (SEQ ID NO:2) and at leastone LM609 grafted light chain polypeptide comprising substantially thesame variable region amino acid sequence as that shown in FIG. 1B (SEQID NO:4) or a functional fragment thereof.
 2. The LM609 grafted antibodyof claim 1 , wherein said functional fragment is selected from the groupconsisting of Fv, Fab, F(ab)₂ and scFV.
 3. A nucleic acid encoding aLM609 grafted heavy chain polypeptide comprising substantially the sameLM609 grafted heavy chain variable region nucleotide sequences as thatshown in FIG. 1A (SEQ ID NO:1) or a fragment thereof.
 4. The nucleicacid of claim 3 , wherein said fragment further comprises a nucleic acidencoding substantially the same nucleotide sequence as the variableregion of said LM609 grafted heavy chain polypeptide (SEQ ID NO:1). 5.The nucleic acid of claim 3 , wherein said fragment further comprises anucleic acid encoding substantially the same nucleotide sequence as aCDR of said LM609 grafted heavy chain polypeptide.
 6. A nucleic acidencoding a LM609 grafted light chain polypeptide comprisingsubstantially the same LM609 grafted light chain variable regionnucleotide sequences as that shown in FIG. 1B (SEQ ID NO:3) or afragment thereof.
 7. The nucleic acid of claim 6 , wherein said fragmentfurther comprises a nucleic acid encoding substantially the samenucleotide sequence as the variable region of said LM609 grafted lightchain polypeptide (SEQ ID NO:3).
 8. The nucleic acid of claim 6 ,wherein said fragment further comprises a nucleic acid encodingsubstantially the same nucleotide sequence as a CDR of said LM609grafted light chain polypeptide.
 9. A nucleic acid encoding a LM609grafted antibody heavy chain polypeptide comprising a nucleotidesequence encoding substantially the same LM609 grafted heavy chainvariable region amino acid sequence as that shown in FIG. 1A (SEQ IDNO:2) or fragment thereof.
 10. The nucleic acid of claim 9 , whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same heavy chain variable region amino acid sequence of said LM609grafted heavy chain amino acid sequence (SEQ ID NO:2).
 11. The nucleicacid of claim 9 , wherein said fragment further comprises a nucleic acidencoding substantially the same heavy chain CDR amino acid sequence ofsaid LM609 grafted heavy chain amino acid sequence.
 12. A nucleic acidencoding a LM609 grafted antibody light chain polypeptide comprising anucleotide sequence encoding substantially the same LM609 grafted lightchain variable region amino acid sequence as that shown in FIG. 1B (SEQID NO:4) or fragment thereof.
 13. The nucleic acid of claim 12 , whereinsaid fragment further comprises a nucleic acid encoding substantiallythe same light chain variable region amino acid sequence of said LM609grafted light chain amino acid sequence (SEQ ID NO:4).
 14. The nucleicacid of claim 12 , wherein said fragment further comprises a nucleicacid encoding substantially the same light chain CDR amino acid sequenceof said LM609 grafted light chain amino acid sequence.
 15. A LM609grafted heavy chain polypeptide comprising substantially the samevariable region amino acid sequence as that shown in FIG. 1A (SEQ IDNO:2) or functional fragment thereof.
 16. The LM609 grafted heavy chainpolypeptide of claim 15 , wherein said functional fragment comprises avariable chain polypeptide or a CDR polypeptide.
 17. A LM609 graftedlight chain polypeptide comprising substantially the same variableregion amino acid sequence as that shown in FIG. 7 (SEQ ID NO:4) or afunctional fragment thereof.
 18. The LM609 grafted light chainpolypeptide of claim 17 , wherein said functional fragment comprises avariable chain polypeptide or a CDR polypeptide.
 19. A method ofinhibiting a function of α_(v)β₃ comprising contacting α_(v)β₃with aLM609 grafted antibody or a functional fragment thereof under conditionswhich allow binding of LM609 grafted antibodies to α_(v)β₃.
 20. Themethod of claim 19 , wherein said functional fragment is selected fromthe group consisting of Fv, Fab, F(ab)₂ and scFV.
 21. The method ofclaim 19 , wherein said function of α_(v)β₃ is binding of α_(v)β₃ to aligand.
 22. The method of claim 19 , wherein said function of α_(v)β₃ isintegrin mediated signal transduction.
 23. A method of treating anα_(v)β₃-mediated disease comprising administering an effective amount ofa LM609 grafted antibody or a functional fragment thereof underconditions which allow binding to α_(v)β₃.
 24. The method of claim 23 ,wherein said functional fragment is selected from the group consistingof Fv, Fab, F(ab)₂ and scFV.
 25. The method of claim 23 , wherein saidα_(v)β₃-mediated disease is angiogenesis or restenosis.